Epithelial to mesenchymal transition (EMT) is a cellular program that converts non-motile epithelial cells into invasive mesenchymal cells. EMT is implicated in cancer metastasis, chemo-resistance, cancer progression, and generation of cancer stem cells (CSCs). Inducing mesenchymal to epithelial transition (MET), the reverse phenomenon of EMT, is proposed as a novel strategy to target triple negative and tamoxifen-resistant breast cancer. Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors, a highly invasive mesenchymal phenotype, and a lack of targeted therapy. Estrogen receptor-positive breast cancer can be targeted by tamoxifen, an ER antagonist. However, these cells undergo EMT over the course of treatment and develop resistance. Thus, there is an urgent need to develop therapeutic interventions to target these aggressive cancers. In this study, we examined the role of novel diphenylamine analogs in converting the mesenchymal phenotype of MDA-MB-231 TNBC cells to a lesser aggressive epithelial phenotype. Using analog-based drug design, a series of diphenylamine analogs were synthesized and initially evaluated for their effect on E-cadherin protein expression and changes incell morphology, which was quantified by measuring the spindle index (SI) value. Selected compound 1 from this series increases the expression of E-cadherin, a primary marker for epithelial cells, and decreases the mesenchymal markers SOX2, ZEB1, Snail, and vimentin. The increase in epithelial markers and the decrease in mesenchymal markers are consistent with a phenotypic switch from spindle-like morphology to cobblestone-like morphology. Furthermore, Compound 1 decreases spheroid viability, cell migration, and cell proliferation in triple negative BT-549 and tamoxifen-resistant MCF-7 breast cancer cells.

Abstract While the precise molecular mechanisms underlying metastasis remain unclear, epithelial-to-mesenchymal transition (EMT), the loss of an epithelial cell phenotype and acquisition of a mesenchymal cell phenotype, has been implicated in cancer cell invasion and dissemination. The ZEB family of transcription factors, which includes ZEB1 and ZEB2, has been demonstrated to mediate this transition by downregulating the expression of genes associated with an epithelial phenotype. We sought to investigate the effects of direct ZEB family overexpression on EMT in estrogen receptor-positive (ER+) breast cancer cell systems. We overexpressed ZEB1 or ZEB2 in the epithelial, ER+, luminal A breast cancer cell lines MCF-7 and ZR75. Overexpression of individual ZEB1 and ZEB2 levels were confirmed and localization of the ZEB factors to the nucleus was confirmed by confocal microscopy in both cell lines. ZEB2 overexpressing cells, but not ZEB1 overexpressing cells, showed increased migration and invasion in vitro compared to the vector control in both MCF-7 and ZR75 cell lines, suggesting differential function of the two ZEB family members. Additionally, MCF-7-ZEB2 xenografts exhibited increased lung metastasis compared to MCF-7-vector cells. To elucidate the effects of ZEB on our ER+ cell line we performed next generation deep sequencing on MCF-7 -vector, ZEB1 and ZEB2 overexpressing cells. Analysis of total gene regulation using the NCI Pathway Interaction Database demonstrated an increase in genes associated with RhoA activity, an important mediator of cell motility, in ZEB2 overexpressing cells. However, the ZEB overexpressing cells show no change in morphology and canonical EMT markers remained unchanged between cell lines, suggesting a potential post-translational modification affecting ZEB1 and ZEB2 function. Together these results indicate that ZEB factors drive motility in breast cancer cells but are incapable of promoting a complete EMT in ER+ cells, warranting further investigation into the mechanisms involved in ZEB action. Elucidating the pathways involved in ZEB family function is an important step in understanding the mechanisms underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay Rhodes, Elizabeth Martin, Theresa Phamduy, Steven Elliot, Van Hoang, Henry Segar, Aaron Buechlein, Douglas Rusch, Dave Miller, Melody Baddoo, Erik Flemington, Kenneth Nephew, Douglas Chrisey, Bridgette Collins-Burow, Matthew Burow. ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2014-1034

CXCR4 is a chemokine receptor often found aberrantly expressed on metastatic tumor cells. To investigate CXCR4 signaling in tumor cell adhesion, we stably overexpressed CXCR4 in MCF7 breast tumor cells. Cell attachment assays demonstrate that stimulation of the receptor with its ligand, CXCL12, promotes adhesion of MCF7-CXCR4 cells to both extracellular matrix and endothelial ligands. To more closely mimic the conditions experienced by a circulating tumor cell, we performed the attachment assays under shear stress conditions. We found that CXCL12-induced tumor cell attachment is much more pronounced under flow. ROCK is a serine/threonine kinase associated with adhesion and metastasis, which is regulated by CXCR4 signaling. Thus, we investigated the contribution of ROCK activity during CXC12-induced adhesion events. Our results demonstrate a biphasic regulation of ROCK in response to adhesion. During the initial attachment, inhibition of ROCK activity is required. Subsequently, re-activation of ROCK activity is required for maturation of adhesion complexes and enhanced tumor cell migration. Interestingly, CXCL12 partially reduces the level of ROCK activity generated by attachment, which supports a model in which stimulation with CXCL12 regulates tumor cell adhesion events by providing an optimal level of ROCK activity for effective migration.

B58 Background: Results of epidemiological studies relating consumption of dietary factors to cancer have increased the knowledge base that provides rationale for various nutritional strategies designed to contribute to cancer prevention and treatment. Of particular interest are the classes of compounds known as the phytoestrogens, which embody several groups of non-steroidal estrogens present in soy containing foods. Phytoestrogens exhibit a number of biological effects, suggesting that they may have a role in cancer prevention. We have identified the phytoestrogens glyceollins I, II and III in soy plants grown under stressed conditions which exhibit marked anti-estrogenic effects on ER function. In this study, we assessed the influence of glyceollin I on MCF-7 breast cancer cells and BG-1 ovarian cancer cells growth and gene expression. Materials and Methods: The effects of glyceollin I on proliferation were determined by colony assays. ER-dependent cells were plated in 6 well plates, treated with glyceollin I with and without estrogen stimulation. Colony formation was determined after 10 days of incubation. Gene expression was determined by ERE-luciferase and real time RT-PCR assays. ER-dependent cancer cell lines were transfected with an ERE-Luc plasmid, treated with glyceollin I in the presence and absence of estrogen stimulation, and harvested for luciferase activity. The cells were also analyzed for the quantitative expression of PgR and SDF-1 genes after glyceollin treatment with and without estrogen stimulation. In addition, binding of glyceollin I to ER-α was demonstrated with a competitive binding assay. Results: We have established the ability of glyceollin I to bind to the ER-α and to inhibit the expression of ER-dependent genes. We also demonstrated the suppression of proliferation on MCF-7 and BG-1 cells [estrogen receptor alpha (ER-α-positive), demonstrating that the effect of glyceollin I is not restricted to the MCF-7 model. Discussion: Our results establish the in vitro inhibition of estrogen-dependent cell growth by glyceollin I and also provide critical information to the understanding of estrogen-related cancers. The glyceollins may represent important components of a soy-based diet in terms of chemoprevention and treatment of estrogen-related cancer.